Summary of Work: Nuclear accumulation of p53 occurs in response to agents causing cell cycle arrest or cell death. However, p53 does appear in cytoplasm as normal cells progress through the cell cycle. In certain tumor cells such as neuro- blastoma which express wild type p53, this protein concentrates in cytosol and cytoplasmic granules. Identification of extranuclear organelles harbor- ing p53 and specific interacting proteins is important to understanding p53 trafficking in normal and diseased cells. We began by studying protein interactions of mutant p53 in human and murine cells. We have identified Hsp proteins which complex and sequester mutant p53 to the cytoplasm and include Grp75, Grp78 and a weakly basic 90 kD protein. Isolation from native lysates has shown cytoplasmic Hsp complexes exist as preformed multimers which may be important for recognition, binding and disposal of aberrantly folded proteins. The presence of Grp75 and Grp78 suggested p53 might be found within these organelles which was confirmed for Grp75 in mitochondria by biochemical and immunoelectron microscopic analyses. Further work shows immunoreactive p53 material is also located on the surface of these cells which may relate to observations of other suggesting degradation of mutant p53 into peptides for antigen presentation. Because Grp75 is a mitochond- rial protein that interacts with p53,we raised Grp75 antibodies to study Grp75 expression after treatment of cells with various Grp-inducing agents. Although overall induction of Grp proteins is modest at 2-3 fold, we found 2-deoxyglucose treatment induced 5 novel Grp75 isoforms in human and murine cells. Appearance of these new Grp75 isoforms appears more indicative of gluose deprivation stress than overall protein induction. Future work might involve study of so-called anchoring proteins in cytoplasm-harbored wildtype p53 expressing tumor cells. A final important aspect of this project is the patented preparative 2D PAGE system developed for isolating and sequencing proteins which was used to identify HSP proteins and others.